Bifunctional molecules

ABSTRACT

A chimeric antibody conjugate comprising an antigen binding region of a non-human antibody and an immunoglobulin constant region which comprises at least one CH domain or epitope thereof, with the proviso that the constant region is not a naturally occurring FC fragment. A bifunctional molecule for use in labelling an antibody derived from a first species, the bifunctional molecule comprising a binding-region which binds to the antibody of the first species or to one or more groups provided thereon, and a constant region derived from an antibody of a second species, the constant region comprising at least one CH domain or an epitope thereof. The present invention relates to bifunctional molecules and complexes which are useful as positive control reagents in antibody based diagnostic tests. The present invention also relates to polynucleotides encoding these bifunctional molecules, and to diagnostic assays involving the use of these molecules.

FIELD OF THE INVENTION

The present invention relates to bifunctional molecules and complexeswhich are useful as a positive control reagents in antibody baseddiagnostic tests. The present invention also relates to polynucleotidesencoding these bifunctional molecules, and to diagnostic assaysinvolving the use of these molecules.

BACKGROUND OF THE INVENTION

Infection of humans by many micro-organisms leads to the initiation of ahumoral immune response that can be used in the diagnosis of thedisease. In the early acute phase of the infection, specific IgM classantibodies are the first to appear in serum 1-4 weeks after the onset ofsymptoms and last for up to three months. IgG class antibodies appearlater and remain elevated throughout the patient's life. Detection of anIgM response is indicative of a recent or current infection, while thepresence of an elevated IgG response is a marker for past exposure tothe causative agent. Specific IgM or IgG responses to a particularinfectious agent can be measured by antibody based diagnostic tests suchas ELISA, immiunochromatography, particle agglutination ELISA, biosensoror other similar assays.

These assays require the use of reactive human sera as a positivecontrol. The positive control reagent is usually serum taken from apatient or animal which is known to have a positive reaction to theparticular antigen under test. If the test is designed to distinguishbetween early and late infection (via the differentiation betweenimmunoreactive IgM, for early infection and IgG, for late or previousinfection), the positive control serum or reagent should containimmunoreactive antibody of the correct immunoglobulin class.

It is becoming increasingly difficult to source sufficient quantities ofimmune human sera or plasma, particularly as diagnostic tests for rarerdiseases become available. Collection of blood for IgM controls frompatients in early stages of infection when clinical symptoms aregenerally most severe poses significant ethical problems, particularlyif the disease primarily affects juveniles. Other drawbacks include therequirement for consistent collections from remote locations, the needto standardise each batch and to check for contamination with infectiousagents such as HIV, hepatitis B and hepatitis C. There are also problemsin obtaining control sera for specific endemic diseases in communitieswhere the donation of blood or blood products is socially unacceptable.

There is therefore a need for a source of positive control reagentswhich does not rely on being obtained from human donors.

Hybridoma technology provides a plentiful supply of monoclonalantibodies, but as these are generally of murine origin, they do reactwith binding reagents used to quantify human antibodies. Intact,functional mouse/human chimeric antibodies have been described in theliterature for some time (Boulianne et al. 1984, Nlorrison et al., 1984;Winter et al. 1991). In these constructs the antigen binding functionresiding in a mouse Fab or Fv fragment has been grafted on to a human Igbackbone and expressed in hebridoma cells. In some cases these reshapedmolecules have been designed for human therapy, utilising the effectorfunctions of the human Fc for targeting (Reichmann et al., 1988). Othershave been designed as positive control reagent substitutes (Hamilton,1990, 1991), where V_(H) and V_(L) regions from a mouse monoclonalantibody of desired specificity have been grafted onto either a humanIgG or IgM backbone.

Synthetic positive control reagents are available from a limited numberof sources. U.S. Pat. No. 4,929,543 relates to chimeric antibodyfragments where Fab or F(ab′)2 fragments of non human origin, withspecificity for the desired antigen, are chemically coupled to human Fcfragments in order to confer upon the reactive non-human Fab fragmentsepitopes recognised by class specific anti human immunoglobulinantisera. This reference does not teach or suggest coupling non-humanFab or F(ab′)2 fragments to individual CH domains in order to provideepitopes for recognition by class specific anti human immunoglobulinantisera. Furthermore, production of the chimeric fragments is entirelyby synthetic routes based upon digestion of antibodies, purification offragments and chemical linking to create the chimera.

Labor Diagnostika GmbH of Heiden, Germany have produced syntheticpositive control reagents which are formed by chemical attachments ofnon human Fab fragments and human Fc fragments onto a latex bead. Theseattachments confer upon the bead the twin properties required of apositive control reagent—specific antigen binding and humanimmunoglobulin class specific epitopes.

A process for producing positive control reagents which circumvents therequirement to manipulate full length Fc fragments, or to manipulate VHand V_(L) sequences for each new control reagent specificity, isdesirable.

SUMMARY OF THE INVENTION

The present inventors have now developed bifunctional molecules whichmay be used as positive control reagents in antibody based diagnostictests.

In one aspect of the present invention, the bifunctional molecule is achimeric antibody conjugate comprising a first region which binds aspecific antigen and a second region comprising at least one constantdomain sequence derived from a class specific immunoglobulin. Thisconjugate, which may be used directly as a positive control reagent,avoids the inconvenience of manipulating full length or naturallyoccurring Fc fragments. Furthermore, the conjugate may be readilyproduced by recombinant DNA technology.

Accordingly, in a first aspect the present invention provides a chimericantibody conjugate comprising an antigen binding region derived from anon-human antibody and a constant region which comprises at least oneC_(H) domain or epitope thereof, with the proviso that the constantregion is not a naturally occurring Fc fragment.

When used herein, “naturally occurring Fc fragment” means a full lengthnaturally occurring Fc fragment which may be derived by proteolyticdigestion of an intact antibody molecule. For example, a naturallyoccurring Fc fragment of IgM will comprise domains C_(H)2, C_(H)3 andC_(H)4, whereas a naturally occurring IgG Fc fragment will compriseC_(H)2 and C_(H)3 domains.

By “chimeric” we mean that the constant region is derived from adifferent species than the antigen binding region.

In a preferred embodiment the non-human antigen binding region comprisesor consists of a non-human Fab fragment or part thereof. The non-humanantigen binding region may comprise or consist of an scFv fragment.

In a further preferred embodiment the non-human antigen binding regionis derived from a mouse.

In a preferred embodiment the constant region is derived from a humanantibody. It will be appreciated, however, that the constant region maybe a non-human (such as bovine, canine, ovine, equine, feline orcaprine) constant region in cases where the chimeric construct is to beused as a positive antibody control in assays involving sera derivedfrom non-human species.

The constant region may consist of a non-naturally occurring combinationof Cal domains or epitopes thereof. The constant region may consist oftwo C_(H) domains of the same type, for example, two C_(H)3 domains.Alternatively, the constant region may consist of two different domains.The two different domains, or epitopes thereof may be derived fromantibodies of different classes. In a preferred embodiment, the constantregion consists of a single C_(H) domain.

In a particularly preferred embodiment of the present invention thechimeric antibody conjugate is suitable as a positive IgM control andthe constant region comprises one or more C_(H)3μ domains.

In a further preferred embodiment the non-human antigen binding regionbinds to an epitope derived from an infectious agent selected from butnot limited to dengue virus, rubella virus, herpes virus, palivovirus,human glycophorin, Rickettsia sibirica, Burlkioleria pseudomallei,Salmonella typhi or paratyphi, Leptospira interrogans, Plasmodiumfalciparum/vivax, Japanese encephalitis virus, Yellow fever virus,Bordetella pertlussis/parapertussis, Candida albicans/kruzei, Varicellazoster virus, HIV, Hepatitis viruses, Human papilloina virus,Epstein-Barr virus, Ross River virus, Brucella aboltis, Humanherpesvirus-6, Parvovirus B19, Coxiella burnettii, Herpes simplexviruses 1&2, Rickettsia rickettsii, Conori australis, Rickettsiatsutsugamuushi.

In a second aspect the present invention provides a recombinantpolynucleotide molecule comprising a sequence encoding a non-human V_(H)region, a sequence encoding a non-human V_(L) region, a sequenceencoding a flexible linker positioned between the V_(H) region sequenceand the V_(L) region sequence, and a heterologous sequence encoding aC_(H) domain or epitope thereof.

By “heterologous sequence encoding a C_(H) domain” we mean sequence,encoding a C_(H) domain which is derived from a different species thanthe sequences encoding the V_(H) and V_(L) regions.

In a preferred embodiment of the second aspect the heterologous sequenceencodes a human CH domain.

By ‘flexible linker’ we mean a region of amino acids of sufficientlength and flexibility to allow the V_(H) and V_(L) polypeptide regionsto dock correctly with respect to each other to form an scFv fragment.The flexible linker may be a polypeptide of between 12 and 30 aminoacids in length. Preferably the linker is a polypeptide of about 15amino acids in length. The linker niav have the sequenceGGGGSGGGGSGGGGS.

In a preferred embodiment, the C_(H) domain sequence is linked to the 3end of the V_(L) or V_(H) sequence. In this preferred construct thechimeric antibody conjugate is synthesized as a single polypeptide chainwhich folds to produce two separate functional domains.

In a further preferred embodiment of the second aspect of the invention,the polynucleotide molecule includes a control sequence which directsthe synthesis of both the V_(L) and V_(H) polypeptide regions. Thecontrol sequence is preferably an inducible promoter such as the lacpromoter.

In a further preferred embodiment the polynucleotide molecule includes asequence encoding a leader peptide which directs the synthesisedpolypeptide chains to the host cell periplasm. The leader sequence maybe the pel B sequence.

In a third aspect the present invention provides a recombinantpolynucleotide molecule comprising a sequence encoding a non-human V_(L)region, a sequence encoding a non-human CL region, a sequence encoding anon-human V_(H) region, a heterologous sequence encoding a C_(H) domainor epitope thereof and optionally a sequence encoding a non-human C_(H)1region.

In a preferred embodiment of the third aspect the heterologous sequenceencodes a human C_(H) domain.

In a further preferred embodiment of the third aspect of the presentinvention, the V_(L) and C_(L) sequences are linked together so that theV_(L) and C_(L) regions are expressed as a single polypeptide. In a morepreferred embodiment, the V_(H) and C_(H)1 sequences are also linkedtogether so that the V_(H) and C_(H)1 regions are expressed as a singlepolypeptide.

In a further preferred embodiment of the third aspect the polynucleotidemolecule includes a control sequence which directs the synthesis of boththe V_(L)-C_(L) and V_(H)-C_(L) polypeptide chains. The control sequenceis preferably an inducible promoter such as the lac promoter.

In a further preferred embodiment of the third aspect the polynucleotidemolecule includes a sequence encoding a leader peptide which directs thesynthesised polypeptide chains to the host cell periplasm. The leadersequence may be the pel B sequence. Preferably, the V_(L)-C_(L) andV_(H)-C_(H)1 polypeptide chains associate in the host cell periplasm andare stabilised by one or more disulphide bonds between the chains.

In a further preferred embodiment of the third aspect the heterologousC_(H) domain sequence is linked to the V_(L)-C_(L) sequences or theV_(H)-C_(H) sequences so that the expressed heterologous C_(H) domain isattached to the V_(L)-C_(L) polypeptide or the V_(H)-C_(H) polypeptide.

In a further preferred embodiment of the third aspect the non-humanC_(H)1 sequence is absent from the recombinant polynucleotide construct.The heterologous C_(H) domain sequence may be linked directly to thenonhuman V_(H) sequence to give rise to a chimeric non human V_(H)-humanC_(H) polypeptide chain. This chimeric polypeptide chain may associatewith the non-human V_(L)-C_(L) polypeptide chain to form a chimeric Fabfragment. It will be appreciated that such a chimeric Fab fragment willpossess a specific antigen binding region, and a human constant regionwhich provides a recognition site for class specific anti immunoglobulinantibodies.

The polynucleotide molecules of the second or third aspects of thepresent invention may be incorporated into plasmids or expressionvectors which may then be introduced into suitable bacterial, yeast,insect or mammalian host cells.

Accordingly, in a fourth aspect the present invention provides a vectorcomprising a polynucleotide according to the second or third aspects ofthe present invention.

In a fifth aspect the present invention provides a bacterial, yeast,insect or mammalian host cell transformed with a vector according to thefourth aspect of the present invention.

In a sixth aspect the present invention provides a method of producing achimeric antibody conjugate which comprises culturing a host cellaccording to the fifth aspect of the present invention under conditionsenabling the expression of the conjugate and optionally recovering theconjugate.

In a seventh aspect the present invention provides a chimeric antibodyconjugate produced by a method according to the sixth aspect of thepresent invention.

In yet another aspect of the present invention the bifunctional moleculeis able to bind to antibodies or antibody-like molecules and therebylabel them with epitopes from immunoglobulin constant regions derivedfrom different species. The complex thus formed has the properties of aspecific positive antibody control: a ligand binding site withspecificity for the antigen, hapten or drug in question and epitopes ordomains which are recognised by immunoglobulin binding reagents. Thebifunctional molecules of this aspect of the invention may be producedby recombinant DNA technology. Alternatively, recombinant fragments maybe linked by conventional chemical coupling technologies.

Accordingly, in an eighth aspect the present invention provides abifunctional molecule for use in labelling an antibody of a firstspecies, the bifunctional molecule comprising a binding region whichbinds to the antibody of the first species or to one or more groupsprovided thereon, and a constant region derived from an antibody of asecond species, the constant region comprising at least one CH₁ domainor an epitope thereof.

The order of the binding and constant regions on the bifunctionalpolypeptide is not critical. The order may be either (Nterminus)—binding region—constant region—(C terminus) or vice versa, ie(N terminus)—constant region—binding region—(C terminus).

In a ninth aspect the present invention provides a complex formedbetween (i) an antibody or biologically active fragment thereof derivedfrom a first species and (ii) a bifunctional molecule, the bifunctionalmolecule comprising a binding region which binds to the antibody of thefirst species or to one or more groups provided thereon, and a constantregion derived from an antibody of a second species, the constant regioncomprising at least one CH domain or an epitope thereof.

By “biologically active fragment” we mean a fragment which mimics thebinding of the antibody derived from the first species to at least oneantigenic determinant.

In a preferred embodiment of the eighth and ninth aspects, the bindingand constant regions of the bifunctional molecule are separated by alinker molecule. The linker molecule may be a short peptide. Preferably,the linker molecule is a peptide of between 1 and 20 amino acids inlength, more preferably between 1 and 10 amino acids in length, and morepreferably between 2 and 5 amino acids in length.

In a further preferred embodiment of the eighth and ninth aspects, thebinding region is not derived from an antibody. By this we mean that thebinding region is preferably not (i) a Fab fragment, (ii) a portion of aFab fragment, (iii) an ScFv fragment or (iv) a portion of an ScFvfragment.

In one embodiment of the eighth and ninth aspects, the binding regionbinds directly to the antibody derived from the first species.

In a further preferred embodiment of the eighth and ninth aspects, thebinding region is derived from a protein selected from the groupconsisting of, Stieptococcal protein G (described in Bjorck and Kronvall(1984), and Boyle and Reis (1987), the entire contents of which areincorporated herein by reference) Staphlylococus aureus protein A(described in Uhlen et al. (1984), and Boyle and Reis (1987), the entirecontents of which are incorporated herein by reference) andPeptostreptococcus magnus protein L (which is described in Åkerstrom andBjörck (1989), the entire contents of which is incorporated herein byreference). In a further preferred emibodiment, the binding regioncomprises one of the immunoglobulin binding regions of Staphylococcusaureus protein A. The immunoglobulin binding region of Staphylococcusaureus protein A may be fragment B.

Iin a further preferred embodiment of the eighth and ninth aspects, thebinding region comprises a mouse Fc γ receptor or fragment thereof. Themouse Fc γ receptor may be selected from the group consisting of FcγRI,which specifically binds monomeric mouse IgG2a; FcγRIII, which bindsaggregated IgG1, IgG2a and IgG2b; and FcγRIII, which binds the minorsubclass IgG3 (see Heusser et al., 1977; Segal et al., 1978; Unkeless etal., 1988; Hogarth et al., 1987; Kulczycki et al., 1990, the entirecontents of which are incorporated herein by reference).

In another preferred embodiment of the eighth and ninth aspects, thebinding region comprises a histidine rich glycoprotein (as described inBorza et al., 1996 and Gorgani et al., 1997, the entire contents ofwhich are incorporated herein by reference).

In another embodiment of the eighth and ninth aspects, the bindingregion binds to one or more groups provided on the antibody of the firstspecies. Preferably, the group(s) is a biotin molecule and the bindingregion comprises streptavidin (described in Argarafia et al. (1986),U.S. Pat. No. 5,672,691 and U.S. Pat. No. 5,489,528, the entire contentsof which are incorporated herein by reference) or a fragment thereof.

In a further preferred embodiment of the eighth and ninth aspects of thepresent invention, the first species is a rat or a mouse.

In a further preferred embodiment of the eighth and ninth aspects, theantibody of the first species is a monoclonal antibody. In a furtherpreferred embodiment, the antibody of the first species is an IgGantibody.

In a further preferred embodiment of the eighth and ninth aspects, theantibody constant region is not a naturally occurring Fc fragment.

In a further preferred embodiment of the eighth and ninth aspects, theantibody constant region comprises or consists of a non-naturallyoccurring combination of iminlunoglobulin C_(H) domains or epitopesthereof. The constant region may include or consist of two CH domains ofthe same type, for example, two C_(H) domains. Alternatively, theconstant region may include or consist of two different domains. The twodifferent domains, or epitopes thereof, may be derived from antibodiesof different classes. In a preferred embodiment, the constant regionconsists of a single C_(H) domain.

In a further preferred embodiment of the eighth and ninth aspects, thesecond species is a human. It will be appreciated, however, that thesecond species may be non-human (for example, bovine, canine, ovine,equine, feline or caprine) in cases where the bifunctional molecule orcomplex is to be used as a positive control reagent in assays involvingsera derived from non-human species.

In a particularly preferred embodiment of the ninth aspect of thepresent invention, the bifunctional molecule is suitable for combinationwith mouse IgG as a positive IgM control and the constant regioncomprises one or more C_(H)3μ domains.

In a particularly preferred embodiment of the ninth aspect of thepresent invention, the bifunctional molecule is bound to a location onthe antibody (or fragment thereof) of the first species which does notsig ificantlyvhinder the binding between the antibody (or fragmentthereof) and its specific antigen.

In a further preferred embodiment of the complex according to the ninthaspect, the affinity between the binding region and the antibody orbiologically active fragment thereof derived from the first species issufficient to form a stable complex in solution. Preferably, the bindingregion has a K_(D) for the antibody of less than 10⁻⁶ M. Miorepreferably, the K_(D) is less than 10⁻⁸ M and more preferably less than10⁻⁹ M.

In a further preferred embodiment of the eighth and ninth aspects, theantibody constant region is modified in order to facilitate theproduction of the molecule, or to reduce aggregation of individualbifunctional molecules, without substantially altering thecharacteristic epitopes of the domain. For example, a cysteine residueusually associated with the formation of an inter-chain disulphide bondmay be mutated to serine. In another example, a bifunctional moleculewhich contains a fragment of Staphylococcal protein A linked to a humanCγ3 domain may aggregate because of the high affinity of the protein Afragment for human IgG constant domains. This aggregation may becircumvented by a substitution His to Arg at position 435. Evidencesuggests that the lack of binding of protein A to human IgG subclass 3is related to the substitution of Arg for His at position 435 (seeDeiseilhofer, 1981, the entire contents of which are incorporated hereinby reference).

It will be appreciated by persons skilled in the art that within thecontext of the present invention, the preferred C_(H) domains orepitopes will be dependent on the intended use of the bifunctionalmolecule. For example, if the bifunctional molecule or complex is to beused as a replacement for positive IgM control sera, the preferred C_(H)domains or epitopes will be C_(H) domains or epitopes. Alternatively, ifthe bifunctional molecule or complex is to be used as a replacement forpositive IgG control sera, the preferred C_(H) domains or epitopes willbe a C_(H) γ domains or epitopes. If the bifunctional molecule orcomplex is to be used as a replacement for positive IgA control sera,the preferred C_(H) domains or epitopes will be a C_(H) α domains orepitopes.

In a tenth aspect, the present invention provides an isolatedpolynucleotide encoding a bifunctional molecule according to the eighthaspect of the present invention.

The polynucleotide molecule of the tenth aspect of the present inventionmay be incorporated into plasmids or expression vectors which may thenbe introduced into suitable bacterial, yeast, insect or mammalian hostcells.

Accordingly, in en eleventh aspect the present invention provides avector comprising a polynucleotide according to the tenth aspect of thepresent invention.

In a twelfth aspect the present invention provides a bacterial, yeast,insect or mammalian host cell transformed with a vector according to theeleventh aspect of the present invention.

In a thirteenth aspect the present invention provides a method ofproducing a bifunctional molecule which comprises culturing a host cellaccording to the twelfth aspect of the present invention underconditions enabling the expression of the bifunctional molecule andoptionally recovering the bifunctional molecule.

In a fourteenth aspect the present invention provides a bifunctionalmolecule produced by a method according to the thirteenth aspect of thepresent invention.

In a fifteenth aspect the present invention provides a method ofproducing a complex according to the ninth aspect which comprisesadmixing an antibody or biologically active fragment thereof derivedfrom a first species with a bifunctional molecule according to theeighth aspect of the present invention.

Methods for detecting antibodies in biological samples are well known.In general, these methods involve incubation of the sample with (i) aantigenic determinant characteristic of a particular disease, and (ii)an anti human Ig antibody. The antibody measurement is generallycompared to a control measurement obtained by incubating the antigenicdeterminant characteristic of the disease and the anti human Ig antibodywith a positive control serum obtained from an individual with thedisease. The present inventors have found that the chimeric antibodyconjugates of the present invention react in diagnostic tests in amanner similar to class specific positive control serum.

Accordingly, in a sixteenth aspect the present invention provides amethod for detecting an antibody in a biological sample which involvescomparing the level of detection obtained with the biological sample tothe level of detection obtained with a positive control, wherein thepositive control comprises a chimeric antibody conjugate according tothe first aspect, or a complex according to the ninth aspect.

In a preferred embodiment of the sixteenth aspect of the presentinvention, the biological sample is a human biological sample.

In a further preferred embodiment of the sixteenth aspect of the presentinvention the antibodies to be detected are antibodies characteristic ofa disease selected from but not limited to dengue fever, Japaneseencephalitis, rubella, spotted fever, herpes infection, parvovirusinfections, melioidosis, typhoid, leptospirosis, malaria, yellow fever,whooping cough, systemic candidiasis/thrush, chicken pox, shingles,AIDS, hepatitis, liver cancer, cervical cancer, infectiousmononucleosis, nasopharvngeal carcinoma, Ross River fever, brucella,exanthum subitum (Sixth disease/Roseola infantum), erythemaingfectiosuin (Fifth disease), Q Fever, cold sores, genital herpes,spotted fever, scrub typhus.

The antibody to be detected in the biological sample may be an antibodyof any class. In a preferred embodiment, however, the antibody is an IgMantibody.

The terms “comprise”, “comprises” and “comprising” as used throughoutthe specification are intended to refer to the inclusion of a statedcomponent or feature or group of components or features with or withoutthe inclusion of a further component or feature or group of componentsor features.

The invention will now be described in detail by reference to thefollowing non-limiting Figures and Examples.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the structure of intact IgG antibody (a) along with the twosub fragments capable of being produced in prokaryotic or lowereukaryotic cells, Fab (b) and scFv (c). The antigen binding region, thelocation of the CDR loops are indicated.

FIG. 2 illustrates one embodiment of the invention. The singlepolypeptide chain protein folds into two domains. The scFv regionderived from mouse DNA sequences folds to form the specific antigenbinding site. The C-domain derived from human DNA sequences ofimmunoglobulin constant regions folds to provide binding epitopes forheterologous, class specific anti human immunoglobulin sera.

FIG. 3 shows in cartoon form the two region s of the bifunctionalbinding molecule and illustrates one embodiment of the invention. Alsoshown is the complex formed between the bifunctional molecule and intactmouse IgG in which the mouse IgG is decorated with antibody C domains,preferably from human immunoglobulin heavy chains.

FIG. 4 shows a further embodiment of the invention in which a complex isformed between the bifunctional molecule and one or more groupsintroduced onto the antibody of species A. In the embodimentillustrated, the group is biotin, the first region which binds to thegroup is streptavidin or a fragment thereof and the antibody is mouseIgG.

FIG. 5 illustrates in cartoon form the structure of the bacterialexpression plasinid containing the sequence of one embodiment of theinvention. The expression cassette contains a chemically induciblepromoter, lac, followed by sequence encoding the components of thechimeric protein. The pel B sequence directs the synthesised protein tothe cell periplasm, the V_(H) and V_(L) regions joined by linkersequence are the mouse scFv antibody, and C_(H) is the humanimmunoglobulin constant domain sequence inserted between the Not I andSac II sites. FLAG is an octapeptide recognition sequence recognised bythe antibody anti FLAG® N12 (Eastman Kodak Co., New Haven, Conn.) usedfor monitoring expression; stop designates a stop codon and TTdesignates a transcription terminator. The remainder of the vector isderived from vector pUC19 (Yaniisch-Perron et al., 1985). The entirevector minus the specific inserts is denoted pGC (Coia et al., 1996)

FIG. 6 illustrates the reactivity in ELISA of the 4 different human IgMC_(H) domain constructs linked to the scFv 1C3 (anti-glycophorin). Withglycophorin bound to the ELISA well, samples containing the gene productwere introduced and incubated for 1 hour at RT. After extensive washing,wells were probed with polyclonal anti human IgM antiserum raised insheep and labelled with horseradish peroxidase. After 1 hour incubationat RT and extensive washing, ABTS was added for colour development whichwas read at 405 nm after 20 mins.

FIG. 7 illustrates the reactivity in a dengue IgM capture ELISA ofunfractionated periplasm taken from an expression culture of the 13C11(anti dengue) scFv-C_(H)3μ domain construct. Positive, negative andcalibrator controls were supplied with the test kit (see text) and usedas directed: (100 μl per assay) at a dilution of 1/100 in serum diluent.Other reagents were as supplied with the kit. Periplasm was diluted ⅕,1/10, 1/50 and 1/100. 100 μl of each dilution including undilutedperiplasm were added to test wells on the ELISA plate, covered andincubated for 1 hour at 370C. After 4 washes with diluted wash buffer,100 μl of a combined mixture of stablized dengue virus antigens withperoxidase labelled anti-denigue monoclonal antibody were added and theplate further incubated for 1 hour at 37° C. After 6 washes with dilutedwash buffer, 100 μl TMB solution was added and colour developmentproceeded for 10 mins. 100 μl Stop solution was then added and thecolour read at 450 nm.

Negative, Calibrator and Positive refer to serum controls supplied withthe test. Peri, peri ⅕-peri 1/100 refer to test samples c6 ntaining the13C11 (anti dengue) scFv-C_(H)3μ domain construct. Peri neg control isderived from a culture of 1C3(antiglycophorin)-C_(H)3μ domain chimera(Example 1).

FIG. 8 shows the DNA sequence of the expression cassette (from pGCvector) containing fragment B from Staphylococcus aureus Protein Ajoined via a short linker to the human IgM heavy chain C_(H)3μ domain,together with a terminal FLAG® tag sequence, pel B leader and trp Aterminator.

FIG. 9 shows in cartoon form the constituents of the ELISA used todemonstrate the binding of various mouse IgG subclasses to thebifunctional linking reagent described in Example 1 which has bound toiminobilised polyclonal anti human IgM antibody, raised in sheep.

FIG. 10 shows the verified sequence of an expression cassette in pGCcomprising the pel B leader sequence, core streptavidin, human IgMC_(H)3 domain and FLAG® tag.

FIG. 11 shows results from size exclusion chromatography on Superdex200of a sample containing refolded Streptavidin-C_(H)3μ in phosphatebuffered saline. Flow rate was 0.5 ml/min.

DETAILED DISCLOSURE OF THE INVENTION

In one aspect the present invention relates to a recombinant chimericantibody molecule. One region of this chimeric molecule comprises anantigen binding region derived from an antibody.

Fragments of antibody molecules containing predominantly antigen bindingregions have been synthesized using prokaryote or lower eukaryoteexpression systems (eg bacterial or yeast cells) (see, for example,PCT/AU93/00491, the entire disclosure of which is incorporated herein byreference). The antigen binding site is composed of amino acid residuesformed in up to six surface loops at the extremity of the molecule. Theloops on the outer domain are termed complementarity determining regions(CDRs) and provide the specificity of binding of the antibody to theantigenic target, by variation in the amino acid composition of thesesurface loops. The antigen binding regions of both intact IgG andsub-fragments are illustrated in FIG. 1.

In order to stabilise the paired associations of the V_(H) and V_(L)regions produced by such cultures, these regions may be expressed as onecontinuous polypeptide chain where there is a region of amino acids ofsufficient length and flexibility interspersed between the C-terminus ofone domain and the N-terminus of the other to allow the two domains todock correctly with each other to correctly position the CDR loops.Methods of manufacture of covalently linked single chain Fv fragmentsare disclosed in U.S. Pat. No. 4,946,778. U.S. Pat. No. 5,132,405 and WO94/07921 the entire contents of which are incorporated herein byreference.

Alternatively, the antigen binding domains can be produced as Fabfragments where two polypeptide chains Vet —C_(H)1 and V_(L)-C_(L) aresynthesised separately froni mouse gene sequences and the subsequentformation of heavy and light chain fragment pairs are stabilised by adisulphide bond between the two chains. (See, for example, Better etal., 1988, Skerra, 1993, Dolezal et al., 1995, the entire contents ofwhich are incorporated herein by reference). A preferred source ofpaired V_(H) and V_(L) genes for the formation of antigen bindingdomains is cDNA prepared from mRNA isolated from mouse monoclonalantibody cell lines.

In one preferred embodiment the chimeric antibody conjugate is apolypeptide chain which, when expressed in E. coli, yeast or mammaliancells from a single gene construction, folds to produce two separatefunctional domains, as shown in FIG. 2. The first domain binds aspecific antigen, and the second domain contains a specificimmunoglobulin constant domain sequence (epitope) which may berecognised and bound by antibodies, prepared in rabbits, sheep or othersuch animal, by imniunisation with class specific human immunoglobulins.

The first functional domain may consist of an antigen binding domain,formed by paired antibody V_(H) and V_(L) regions either a) linked inactive conformation via a flexible peptide linker as in a scFv moleculeor b) linked to mouse Cl₁ and CL domains as in a Fab antibody fragment.The flexible linker used to link the V_(H) and V_(L) regions as in ascFv molecule may be a polypeptide of between 12 and 30 amino acids inlength (Hustonet al., 1991). The V_(H) and V_(L) gene sequences whichcode for the antibody V_(H) and V_(L) regions may be amplified via PCRfrom cDNA of non-human origin (usually prepared from a mouse monoclonalantibody cell line producing antibody with binding specificity for theantigen being assayed in the diagnostic test in question). Any antigenbinding specificity may be incorporated in this domain in either the Fabor scFv conformation. Preferably, a mouse monoclonal antibody cell lineexists with that specificity or a V_(H)/V_(L) pair has been selectedfrom an antibody phage library with binding specificity for thatparticular antigen.

The second functional domain may consist of either a single heavy chainconstant domain or several in tandem which display binding sites(epitopes) for class specific polyclonal anti: immuunoglobtilinantisera, also known as capture antibodies. The C_(H) region genesequences may be amplified via PCR from cDNA prepared from mRNA isolatedfrom peripheral blood lymphocytes. The C_(H) regions can be from any ofthe immunoglobulin heavy chain genes, (those for IgM, IgG, IgA, IgD,IgE) and the gene product from the particular C_(H) region is bound bythe class specific anti immunoglobulin antiserum.

It will be appreciated that chimeric antibody conjugates of the presentinvention are capable of reacting in diagnostic tests in a mannersimilar to a class specific positive control serum. The chimericconjugate will bind to a specific antigen, and will in turn be bound bythe class specific capture antibodies which determine itsimmunoglobulin. An advantage of the conjugates of the present inventionis that they may be produced in large quantities, free of contaminants,by recombinant DNA technology.

In yet another aspect the present invention relates to a bifunctionalmolecule which is able to bind to antibodies or antibody-like moleculesand thereby label them with epitopes from immunoglobulin constantregions derived from different species. Preferably, the bifunctionalmolecule is a single polypeptide chain which when expressed in E. coli,yeast or mammalian cells folds to produce two separate functionaldomains, as shown in FIG. 3. The first domain preferably binds to aspecific region of an antibody, for example, mouse IgG, and the seconddomain contains a specific immunoglobulin constant domain sequence(epitope) which may be recognised and bound by antibodies, prepared inrabbits, sheep or other such animal, by immunisatioin with classspecific human immunoglobulins.

The affinity of binding between the binding region and antibody ispreferably sufficient to form a stable complex in solution between mouseIgG and the bifunctional molecule. The region on the antibody to whichthe bifunctional molecule binds is preferably in a location which willnot sterically hinder the binding between the mouse IgG antibody bindingsite and its specific antigen.

In one embodiment, the binding region binds to a group provided on theantibody. This particular embodiment is illustrated in FIG. 4, in whichthe binding region comprises streptavidin or a fragment thereof and theantibody is mouse IgG.

The invention will be described in detail by reference to the followinignon-limiting examples.

EXAMPLE 1

Production of a C-Domain (IgM) Extended scFv

The gene sequences of the four constant domains (C domains) of human IgMheavy chain were separately amplified from cDNA prepared from mRNAisolated from human peripheral blood lymphocytes using polymerase chainreaction techniques. The design of the oligonucleotide primers used inthe amplifications was based upon the 5′ and 3′ base sequence of each ofthe four IgM heavy chain exons, obtained through GENBANK accessionX14940 (Dorai and Gillies, 1989).

In the primers, specific restriction enzyme recognition sequences wereadded (NotI at the 5′ end and SacII at the 3′ end) to each exon sequenceto facilitate the introduction of the C domain sequence at a specificsite in a previously constructed plasmid expression vector. Theexpression cassette of this E. coli plasmid vector (pGC; Coia et al.,1996) contained V_(H) and V_(L) sequences from the mouse monoclonalantibody cell line 1C3, (Rylatt et al., 1990, WO91/04492) with bindingspecificity for human glycophorin. These were arrayed in the scFvformat, with the 3′ end of the V_(H) sequence linked to the V_(L)sequence via a 45 bp sequence which coded for the protein sequenceGGGGSGGGGSGGGGS. In the synthesized protein, this flexible linker regionallows the correct docking of V_(H) with V_(L). The site for theintroduction of the heavy chain exon was at the 3′ end of the mouseV_(L) sequence without any intervening sequence, save that for therestriction sites, as shown in FIG. 5. Fragments were ligated togetherusing the normal protocols and the ligation mix used to transform E.coli strain XL1-Blue by electroporation.

Recombinant protein was produced from positively transformed colonies ofthe four different constructions (each with a different IgM C-domainsequence) by induction of the plasmid lac promoter with 0.5 mM IPTG inlog phase cultures grown at 30° C. After a further 4 hours incubation at25° C., the cell pellet was harvested by centrifugation, and thecontents of the cell periplasin isolated using the protocol of Minsky etal. (1986).

The periplasmic fraction was assayed by ELISA for the presence ofprotein molecules with the following properties —1) the ability to bindto immobilised glvcophorin on the ELISA plate and 2) a target for thebinding of horseradish peroxidase-labelled polyclonal anti human IgMserum (prepared in sheep. Silenus Laboratories. Melbourne Australia).The results in FIG. 6 show that the construction with IgNI constantdomain 3 (C_(H)3μ) gave the strongest reaction with the labelledpolyclonal antiserum to human IgNi. This was followed by domain C_(H)2μ,with domains C_(H)4μ and C_(H)1μ showing the weakest reactions. It was asurprising observation that the majority of the reactivity of thepolyclonal antiserum was directed to one domain of the IgM heavy chain.Thus, for this particular polyclonal antiserum directed against humanIgM, the construct scFv-C_(H)3μ is a molecule with the preferredproperties of a substitute for a positive human serum control.

A scFv with binding specificity for human glycophorin was solely used todemonstrate the present invention. As will be appreciated by personsskilled in this field, the antigen binding portion of the conjugate maybe replaced with binding specificities to other antigenic entities whichare the focus of a wide range of diagnostic test applications.

EXAMPLE 2 Construction of Extended scFv (13C11 Antidengue) Linked to aHuman IgM C Domain

The reagent was produced from a DNA construct in which the coding regionfor a mouse scFv directed against dengue virus was genetically linked tothat of the third constant domain of human IgM heavy chain (C_(H)3μ),cDNA was prepared from mRNA isolated from the mouse monoclonal antibodycell line 13C11, which specificity for Dengue virus surface antigens(Queensland University of Technology and PanBio Pty Ltd.).Immunoglobulin V_(H) and V_(L) domain DNA sequences were amplified fromthe cDNA using polymerase chain reaction and oligonucleotide primer setsaccording to Zhou et al. (1994). These were linked in the scFv formatV_(H)-linker-V_(L), where the linker was a 45 bp nucleic acid sequencecoding for the protein sequence GGGGSGGGGSGGGGSGGGGS. The resultantfragment was digested with restriction endonucleases Nco I and Not I andpurified by agarose gel electrophoresis. The expression vector asdescribed in Example 1 which contained the 1C3 (antiglycophorin)scFv-C_(H)3μ domain sequence was also digested with Nco I and Not I toexcise the coding sequence for the 1C3 scFv. The remainder of the vector(plus C domain coding sequence) was purified and ligated with thedigested 13C11 (anti dengue) scFv coding region uising standardprotocols. This ligated DNA was then used to transform E. coli strainXL1-Blue by electroporation. Positive colonies were verified both by DNAsequencing and small scale protein expression.

E. coli strain TOPP6 (Stratagene. La Jolla, Calif.) was transformed withpurified plasmid DNA from a verified clone and used for proteinexpression. 500 ml of 2×YT medium (1.0% Yeast extract, 1.6% BactoTryptone. 1.0% NaCl) supplemented with ampicillin at 200-Lg/ml wasinoculated with an overnight culture of the transformed TOPP6 cells andincubated at 370C with agitation until the A₆₀₀ had reached 1.0. IPTG(isopropylthiogalactoside) was added to a concentration of 0.5 mM toinduce the expression of the chimeric gene construct. The culture wasshaken for a further 4 hours at a 25° C. Cells were harvested bycentrifugation and the periplasmic contents isolated using the protocolof Minsky et al. (1986).

Dilutions of the unfractionated periplasm were analysed in the DengueIgM Capture ELISA kit (PanBio Ltd, Windsor, QLD, Australia: Sang et al.,1998) using positive, negative and calibrator serum controls as suppliedin the kit. In the assay, human IgM antibodies are captured by surfacebound polyclonal anti human IgM antiserum (Silenus), and incubated withsoluble dengue antigens plus a peroxidase labelled, dengueantigen-specific monoclonal antibody, to reveal the presence of denguespecific antibodies.

The results are shown in FIG. 7. The periplasm fractions reactedpositively, with the neat, ⅕ and 1/10 dilutions giving higher absorbancereadings at 450 nm than the normal control. At 1/50, the periplasm stillgave an absorbance reading greater than the calibrator control, whichmarks the cut-off between positive and negative reactions. It wascalculated that an absorbance reading equivalent to the positive controlwould have been obtained from a periplasnm dilution of 1/30. Periplasmfrom a culture of an unrelated chimera (1C3-C_(H)3μ, anti-glycophorin,see Example 1) showed no positive reaction in this test.

EXAMPLE 3 Construction of Extended scFv (13C11 Anti Dengue) Linked to aHuman IgG C-Domain

The gene sequences of human IgG constant domains 2 and 3 were separatelyamplified from cDNA from mRNA isolated from human peripheral bloodlymphocytes using polymerase chain reaction techniques. The design ofthe oligonucleotide primers used in the amplifications were based uponthe 5′ and 3′ sequences for each of the heavy chain exons obtainedthrough Genbank accession no E06998.

Sequences coding for NotI and SacII restriction sites were added to the5′ and 3′ end respectively of the C_(H)2γ and C_(H)3γ sequences toenable the insertion into pGC 13C11-C_(H)3μ from which the C_(H)3μsequence had been removed as a NotI-SacIh fragment.

Expression in E. coli and purification of product was performed asdescribed in Example 2. The presence of product in the periplasmicfraction was confirmed by analysis of samples by polyacrylamide gelelectrophoresis and Western blotting, probing the FLAG® tag using mouseanti FLAG® M2 antibody (Hopp et al., 1988) The products were denoted13C11q2y and 13C11C_(H)3γ respectively.

PanBio Indirect Dengue ELISA using 13C11C_(H)2γ and 13C11CH3γ

Periplasmic samples containing 13C11C_(H)2γ and 13C11C_(H)3γ werediluted 1/10 in PBS/0.05% Tween 20 and 100 μl loaded in duplicate on anELISA plate coated with dengue antigens (PanBio Dengue Indirect ELISAKit Cat DET500) and incubated for 1 hour at 37° C. Controls includedPBS/Tween 20 as negative control and positive IgG control serum whichwas probed with both anti human IgM and IgG antibodies. After 6 washeswith PBS/0.05% Tween20, samples and controls were then probed witheither sheep anti human IgM or IgG labelled with horseradish peroxidase(Silenus/AMRAD, Melbourne) at 1:1000 dilution in PBS/0.05% Tween20. Theplate was incubated at 37° C. for 1 hour then washed 6 times aspreviously. 100 μl TMB reagent, supplied with the kit, was added to eachwell, the plate incubated at room temperature for 10 min. then thereaction was stopped by the addition of 100 μl 1M phosphoric acid.Colour intensity was read at 450 nm. The results are shown in Table 1.TABLE 1 Absorbance 450 nm HRP-anti HRP-anti Human Human IgG IgM 13C11CH3γ Periplasm 1/10 1.684, 1.670 0.111, 0.109 13C11 CH2γ Periplasm 1/100.500, 0.586 0.133, 0.113 Human IgG (Dengue Positive) 1/100 0.971, 0.9400.202, 0.361 PBS/0.05% Tween 20 0.308, 0.261 0.112, 0.275

Both 13C11C_(H)3γ and 13C11C_(H)2γ extended scFvs show a positivereaction above background in this ELISA, the results with the 13C11C_(H)3γ periplasin being comparable or better than the Human IgG denguepositive control. The reaction with the construct containing the humanIgG C_(H)3γ domain gave a stronger response than with the C_(H)2γ domainindicating that extended scFvs with a C_(H)3γ domain would be thepreferred construct for use as a replacement IgG positive control.

EXAMPLE 4 Production of a Bifunctional Molecule Containing the BFragment of Staphylococcal Protein A Linked to a Human IgM C-Domain(FB-C_(H)3μ)

The gene sequence for fragment B of Protein A (FB) from Staphylococcusaureus was amplified from chromosomal DNA prepared from strain ATCC2592.3 using polymerase chain reaction techniques. The design of theoligonucleotide primers used in the amplification was based upon the 5′and 3′ base sequences as reported by Uhlen et al. (1984), also GENBANKaccession J01786. In the primers specific restriction enzyme recognitionsites were added NcoI at the 5′ end and NotI at the 3′ end to facilitatethe introduction at a specific site in a previously constructed plasmidexpression vector (pGC; Coia et al., 1996).

In this vector the sequence encoding a human IgNI C domain (C_(H)3μ) hadpreviously been inserted as a NotI-SacII fragment. A short sequenceencoding the three, amino acids Ser, Asp, Pro was included downstream ofthe FB fragment and before the Not I site to introduce some flexibilitybetween the FB domain and the human C_(H)3μ domain. The HumanC_(H)3μdomain had previously been amplified from cDNA prepared from mRNAisolated from human peripheral blood lymphocytes using polymerase chainreaction techniques, using oligonucleotides based upon the 5′ and 3′sequences of the domain obtained through GENBANK accession X14940 (Doraiand Gillies, 1989). We have demonstrated herein that human IgM C domain3 (C_(H)3μ) contains the major reactive epitopes which are bound byseveral polyclonal and monoclonal anti human Iglo antisera capturereagents. Fragments were ligated together using standard ligationprotocols and the ligation mix then used to transformi E. coli strainXL1 Blue by electroporation. The complete DNA sequence of the expressioncassette comprising the pel B leader sequence, fragment B of S. aureusPriotein A, human C_(H)3μ domain, and FLAG® (a tag recognition sequence,Hopp et al., 1988) was verified by using automatic DNA sequencingmethods and is shown in SEQ ID NO: 2 and FIG. 8.

Recombinant protein was produced from positively transformed E. colicolonies by induction of the lac promoter with 0.2 mM IPTG (isopropylβ-d-thio galactoside) in log phase cultures grown at 37° C. Cultureswere induced at a A₆₀₀ of 1.5-2 and incubated for a further 16 hours at18° C. The cell pellet was then harvested by centrifugation and thecontents of the cell periplasm isolated using the protocol of Minsky etal. (1986).

Analysis of the periplasmic fraction by polyacrylamide gelelectrophoresis and Western blot probed with mouse anti FLAG® N12antibody revealed the presence of a FLAG-tagged component in theperiplasm with an approximate molecular weight (NIr) of 20 ktD. Theperiplasmic fraction was then assayed by ELISA to reveal the presence ofprotein molecules with the following properties:

1. The ability to bind to polyclonal anti human IgM antibody prepared insheep and immobilised on the ELISA.

2. The ability to bind to intact mouse IgG as detected by the additionof goat anti mouse IgG antibody, labelled with horseradish peroxidasewhich reacts with TNB (3′,3′,5′,5′,-tetramethylbenzidine) to produce acoloured product measured at 450 nm.

Property 1 was demonstrated by an ELISA in which crude periplasm wasreacted with immobilised polyclonal anti human IgM capture antibody,then probed with mouse anti FLAG® antibody together with goat anti mouseIgG labelled with horseradish peroxidase to detect the C terminal FLAGtag. ELISA plate wells were coated with polyclonal sheep anti human IgMantiserum (Sang et al., 1998), blocked with 5% Skim milk powder in PBSat 37° C. for 1.5 hours. Between each addition step, the wells werewashed 10 times with PBS-0.05% Tween 20. Each addition (100 μl) wasincubated for 20 min at room temperature. NIouse anti FLAG® (EastmanKodak Co. New Haven. CT) was used at 1 μg/ml in PBS-0.05% Tween 20. Goatanti mouse IgG Fc-HRP was used at 0.16 μg/irn in PBS-0.05% Tween 20.Colour was developed by the addition of 100 μl TNFB reagent(3′,3′,5′,5′,-tetramethylbenzidine plus H₂O₂), incubation at roomtemperature for 10 mins followed by the addition of 100 μM Phosphoricacid, and incubation at room temperature for 10 mins. Wells were thenread at 450 nm in an ELISA micro plate reader. The results are shown inTable 2. TABLE 2 1st addition 2nd addition 3rd addition A450 Periplasmmouse anti Goat anti-mouse Ig >3.000 (4 wells) FLAG ® HRP PBS mouse antiGoat anti-mouse Ig 0.124 ± 0.008 (3 FLAG ® HRP wells) PBS PBS Goatanti-mouse Ig 0.094 HRP

The positive result could arise from a combination of the binding ofanti FLAG via the FLAG epitope, or the binding of the mouse IgG with theFB domain on the bifunctional molecule. Regardless of the proportionalcontributions from either of these reactions, the result demonstratesthat the bifunctional molecule can be captured by anti human IgM captureantibodies.

Property 2 was tested using an ELISA sandwich as shown in FIG. 9. Fourmouse IgG subclasses were each individually tested for their ability tobind to the bifunctional molecule. Reagents were from AlvA, Melbourne,Australia (Mouse IgG1: 12CONT01 batch WD12A; IgG2a: 12CONT02 batchUI17A; IgG2b: 121LA01 batch UK18A; IgG3 Rota Ser4 batch UKO7-B1). Eachwas diluted to 1 μg/ml with PBS/0.05% Tween20 before use. The controllinker reagent FB-C_(H)3μ was diluted serially from 1/20 to 1/320 and100 μl loaded into ELISA wells coated with stabilised sheep anti-humanIgM (PanBio Pty Ltd) and incubated for 1 hour at 37° C. After 6 washeswith PBS/0.05% Tween20 100 μl of each diluted mouse IgG subclass wasadded and the plate incubated a further hour at 37° C. After 6 washes inPBS/0.05% Tween20, 100 μl HRP-labelled goat anti mouse IgG Fc (PierceChemical Co. Rockford, Ill.) was added at 0.16 μg/ml, the plate thenincubated for 1 hour and washed 6 times. The reaction was developed with100 μl TNIB solution for 10 min. stopped by adding 100 μl 1M phosphoricacid and the absorbance read at 450 nm. The results are shown in Table3. TABLE 3 Dilution of Control linker Mouse Mouse Mouse Mouse reagentIgG1 IgG 2a IgG 2b IgG3 FBCH3μ 1 μg/ml 1 μg/ml 1 μg/ml 1 μg/ml 1/20 2.425 1.134 0.762 0.599 1/40  2.007 0.555 0.787 0.489 1/80  2.010 0.7760.578 0.289 1/160 1.429 0.581 0.399 0.373 1/320 1.123 0.320 0.309 0.302PBS 0.260 (av of 3)

These results show that under the conditions of the reaction, thebinding of mouse IgG subclasses to the control linker reagent is rankedin the following order: IgG1, IgG2a, IgG2b, IgG3, from highest tolowest. Control reagents formed using a Staphylococcus protein Afragment B-C domain linker would be most successful if mouse IgG1 isused to form the complex. It will be appreciated by those skilled in theart that if the subclass of the mouse monoclonal antibody is IgG3, afront end domain other than protein A would preferably be used toproduce the bifunctional molecule. Suitable alternatives are describedin the “Summary of the Invention” section of this specification.

The bifunctional molecule was separated from other periplasmiccomponents by affinity chromatography on matrix bound mouse anti FLAG®antibody. The fraction which bound to the column was eluted with 0.1 MGlycine HCl pH 3.0 then adjusted to neutrality with saturated Tris. Thebifunctional molecule (denoted FB-C_(H)31) was concentrated to a finalconcentration of approx 1.2 mg/mil and used in an indirect ELISA test.

Human Herpes Virus 6 (HHV6) Indirect ELISA

Tissue culture supernatant containing mouse monoclonal antibody to HHV6was diluted 1/50 in PBS-0.05% Tween 20 and added to ELISA platespreviously coated with HBV6 antigen and incubated at 37° C. for 30 min.After. 4 washes with PBS-Tween, FB-C_(H)3μ was added to subsequent wellsin doubling dilutions from 1/20 to 1/1280 in similar diluent andincubated a further 30 mins at 37° C. After 4 washes with diluent,polyclonal sheep anti human Iglvi labelled with horseradish peroxidase(AvIAD, Mielbourne, 1/1500) was added and incubated 20 min at 37C. Wellswere washed 6 times with PBS and the peroxidase reaction was developedusing 100 μl TDIB solution (3′,3′,5′,5′-Tetramethylbenzidine; BioChemImmunoSystems Italia SPA) for 10 mills and the reaction stopped by theaddition of 100 μl 1M Phosphoric acid. Results are presented in Table 4.The results demonstrate an effective positive reaction to dilutions asgreat as 1/80. TABLE 4 Dilution of FB-C_(H)3μ A₄₅₀ No FB-C_(H)3μ (zero)0.050 1/20 0.771 1/40 0.512 1/80 0.384  1/160 0.197  1/320 0.139  1/6400.087  1/1280 0.079Use of FB-C_(H)3 μl Control Linker in AMRAD Hepatitis E AntibodyIndirect ELISA

The control linker FB-C_(H)3μ was mixed with mouse IgG1 monoclonalantibody to the conformational epitope of Hepatitis E virus (Ref code2E2) and used in an indirect ELISA test, comnparing the response topositive and negative serum controls provided with the test kit (AMRAD,Melbourne, Vic). The control linker sample was partially purified andconcentrated from material located in the periplasmic fraction. Thecontrol linker and mouse HEV antibody were mixed prior to the assay suchthat there was a dilution series of mouse monoclonal antibody from 0 to50 μg/ml at control reagent dilutions of 1:10 and 1:50. The humanpositive control was seriallv diluted from 1/200 and the negativecontrol diluted 1/200 with serum diluent supplied with the kit. Sampleswere added to an ELISA plate (A1NRAD hepatitis E virus coated platesbatch #1401H037) and incubated at room temperature for 30 min. After 3washes with PBS/0.05% Tween20, 100 μl anti-human IgM-HRP conjugate(Silenus; 1:10,000) was added, incubated a further 30 min at roomtemperature, washed 3 times and TNIB substrate added. After 10 minincubation, the reaction was stopped with 1M sulphuric acid and theplate read at 450 nm. The results are shown in Table 5. TABLE 5 Controlreagent Positive Control 2E2 Mab Dilution Dilution Series conc (μg/ml)1:10 1:50 Dilution A450 50 2.92 1.828 1/200  2.745 25 2.931 1.795 1/400 2.135 10 2.880 1.772 1/800  1.525 5 2.900 1.703 1/1600 0.799 2 2.3781.312 1/3200 0.505 1 2.112 0.792 1/6400 0.296 0 0.025 0.023  1/128000.168 Negative Control 0.103 1/200 

These results show that Premixed FB-C_(H)3μ control linker/mousemonoclonal antibody can serve as a suitable positive IgM1 control in theAMRAD HEV ELISA assay.

Levels which give comparable A450 to serum controls are:

-   -   Control Linker 1/10+Mab 2 μg/ml    -   Control Linker 1/50+Mab 50 μg/ml.

No significant background problems are observed indicating that this isa viable option to serum controls in the HEV assay.

EXAMPLE 5 Production of a Bifunctional Molecule Containing the BFragment of Staphylococcal Protein A linked to a Human IgG C-Domain

The gene sequences of human IgG constant domains 2 and 3 were separatelyamplified from cDNA prepared from imNA isolated from human peripheralblood lymphocytes using polymerase chain reaction techniques. The designof the oligonucleotide primers used in the amplifications was based uponthe 5′ and 3′ sequences for each of the heavy chain exons, obtainedthrough Genibank accessioni no E06998.

Whereas Staphylococcal protein A (SPA) exhibits a stronger affinity forhuman IgG1, 2 and 4 than for mouse IgG subclasses, binding to human IgG3is negligible (Reis et al, 1984). It has been suggested that thesubstitution of histidine with arginine at position 435 in IgG3 preventsthe binding to Protein A (Deisenhofer, 1981). Therefore in order tominimise any self aggregation of a bifunctional construct betweenFragment B of SPA and human IgG C domains, it would be preferable tohave any C3γ domain sequence contain the IgG3 mutation, Ar. It is notpossible to selectively amplify IgG3 constant region sequences from cDNAbecause of the close homology of the 5′ and 3′ terminal sequencesbetween all human IgG subclasses. Consequently the mutation wasperformed subsequent to the amplification and cloning using theQuikChange™ Site Directed mutagenesis kit (Stratagene, La Jolla,Calif.).

Sequences coding for NotI and SacI sites were added to the 5′ and 3′ endrespectively of C_(H)2γ and C_(H)3γ sequences to enable insertion intothe expression vector pGC FB-C_(H)3μ, shown in FIG. 8, from which theC_(H)3μ sequence was removed as a NotI-SaclI fragment.

Expression in E. coli and purification of product was performed asdescribed in Example 4.

PanBio IgG Indirect Dengue ELISA using FB-C_(H)2γ and FB-C_(H)3γ ControlLinkers

Test samples were unfractionated periplasmic fractions containingFB-C_(H)2γ and FB-C_(H)3γ control linkers from 500 ml expressioncultures. Samples were used neat or diluted 1:10 in PBS/0.05% Tween 20.

Mouse anti dengue monoclonal antibody was clone 13C11 (IgG2a) obtainedfrom PanBio Ltd (Windsor, Qld) at 1.6 mg/ml and used at a finalconcentration of 1.6 μg/ml diluted in PBS/0.05% Tween 20.

The human positive serum control containing anti dengue IgG antibodieswas obtained from PanBio Ltd and is identical to what is supplied intheir commercial dengue ELISA test. It was used at a dilution of 1:100in PBS/0.05% Tween 20.

HRP-labelled sheep anti human IgG (lot TJ19B) was from Silenus/AMv1RAD(Melbourne) and used at a dilution of 1:1000 in PBS/0.05% Tween 20.

The ELISA plate coated with dengue antigens was as supplied by PanBio intheir commercial Dengue ELISA test. It was used without furtherblocking. All incubations were for 1 hour at 37° C. followed by 3×2 minwashes with PBS/0.05% Tween 20.

The first layer of the ELISA contained 100 μl 13C11 mouse anti denguemonoclonal antibody; control wells contained PBS/0.05% Tween 20.Following incubation and washing as described the samples containingFB-C_(H)2μ and FB-C_(H)3γ were added. Controls contained either humananti dengue IgG serum 1:100 or PBS/0.05% Tween 20. Following incubationand washing, HRP-labelled sheep anti human IgG 1:1000 was added. Afterincubation and washing, 100 μl TMB solution (containing H₂O₂) was addedand incubated for 10 min at room temperature to develop the colourreaction. 100 μl M phosphoric acid was added to stop the reaction andthe plate read in a micro plate reader at 450 nm. Results are shown inTable 6. TABLE 6 A450 13C11mAb + FB-C_(H)3γ periplasm 1.475 1.220PBS-Tween + FB-C_(H)3γ periplasm (control) 0.564 13C11mAb + FB-C_(H)3γperiplasm 1:10 0.901 0.825 PBS-Tween + FB-C_(H)3γ periplasm 1:10(control) 0.268 13C11mAb + FB-C_(H)2γ periplasm 0.856 0.814 PBS-Tween +FB-C_(H)2γ periplasm (control) 0.411 13C11mAb + FB-C_(H)2γ periplasm1:10 0.545 0.521 PBS-Tween + FB-C_(H)2γ periplasm 1:10 (control) 0.279PBS-Tween + human anti dengue IgG positive 0.930 0.922 control 1:100PBS-Tween + PBS-Tween 0.276 0.265

Both periplasmic fractions containing FB-C_(H)2γ and FB-C_(H)3γ providepositive reactions in this ELISA when linked with the mouse anti denguemAb, 13C11, compared to controls. The linker containing the C_(H)3γdomain is the preferred construct to mix with a specific mousemonoclonal antibody to use as a replacement IgG positive controlreagent.

EXAMPLE 6 Bifunctional Construct Using Core Streptavidin as the IgBinding Domain

The protein streptavidin produced by Streptomyces sp. has an affinity(K_(D)) for biotin of the order of 10⁻¹⁵ M (Green, 1975; Paihler et al.,1987) Commercially produced streptavidin consists of a N- andC-terminally shortened form, called core streptavidin (Argarana et al.,1986) comprising the sequence from Ala¹³ or Glu¹⁴ to Ala¹³⁸ to Ser¹³⁹ ofthe mature polypeptide. Core streptavidin is more soluble than the fulllength protein aand its binding activity for biotinylated proteins issignificantly enhanced (Bayer et al., 1989).

The nucleotide sequence for the intact streptavidin gene fromStreptomyces avidinii was obtained from Genbank accession no. X03591(Argaraña et al., 1986).

The structural gene encoding core streptavidin was amplified fromchromosomal DNA of S. avidinii (ATCC27419) using Pfu DNA polymerase andoligonucleotides able to recognise the 5′ and 3′ sequences of the corestreptavidin (codons from Ala¹³ to Ser¹³⁹). The oligonucleotide primersalso contained sequences flanking the 5′ and 3′ streptavidin sequencesfor restriction sites (in particular NcoI at the 5′ end and NotI at the3′ end to enable the core streptavidin gene to be inserted into thevector pGC (Coia et al., 1996) which already contains the sequence forthe human IgNI C_(H)3 domain, in the configuration streptavidin-Cdomain. The sequence coding for the FLAG® tag epitope (Hopp et al.,1998) lies 3′ to the C domain to enable the FLAG® tag to be expressed asa C-terminal peptide on the molecule.

The amplified core streptavidin gene was inserted into the PCR-Script™SK(+) plasmid using the PCR-Script™ Cloning Kit obtained fromStratagene, La Jolla. Calif. (Cat no. 211190-5). After the DNA sequencewas confirmed in positive transformants, the core streptavidin sequencewas excised from the plasmid by double digestion with NcoI and NotI, andligated into a likewise digested pGC vector containing the DNA sequencefor human IgM C_(H)3 domain.

The verified sequence of the expression cassette in pGC comprising thepel B leader sequence, core streptavidin, human IgM C_(H)3 domain andFLAGS tag is shown in SEQ ID NO: 4 and FIG. 10.

Expression in E. coli was performed as described in Example 4. Cellsfrom a 500 ml culture were fractionated into periplasmic fraction,cytoplasmic and membrane fraction. The periplasmic fraction was preparedusing the protocol of Minsky et al. (1986). The cell pellet remainingafter centrifugation to obtain the periplasmic supernatant wasresuspended in TE buffer (10 mM Tris HCl pH 7.4, 1 mM EDTA) sonicatedand centrifuged at 20,000×g to obtain the soluble cytoplasmic fractionand the membrane pellet. Western blot analysis of each of the threefractions using the FLAGS tag as a probe indicated that while theexpressed product was present in all three fractions, the membranepellet contained the highest levels.

The membrane fraction was dissolved in 10 ml 6M guanidinium HCl, pH 1.5(Schmidt and Skerra, 1994), dialysed twice against 200 ml 6N1guanidinium HCl, pH 1.5, then twice against 2L PBS at 4° C. Aftercentrifugation to remove insoluble aggregate, the supernatant wasfractionated on a Superdex 200 (HR 10/30, Pharmacia LKB Biotechnology)column run in PBS at 0.5 ml/niin. The elution profile is shown in FIG.11. All three peaks probed with FLAG® indicating the presence of theproduct. The first peak is high molecular weight aggregate eluting atthe void volume of the column. The second and third peaks were collectedseparately and labelled preparation B (0.10 mg/ml) and preparation A(0.17 mg/ml) respectively. Both preparations showed bands on Westernblot of identical size, and so the size difference between the twopreparations is related to the multimerisation state of the product.Both preparations were used in tests as described below.

Use of Control Reagent Streptavidin-C_(H)3μ in PanBio Dengue IndirectELISA

In this test, a complex is formed between biotinylated monoclonal mouseIgG to dengue antigens (13C11-B) and streptavidin linked to human IgMC_(H)3 domain (strep-C₁₋₃p) to mimic positive human IgM antibody todengue, and used as a pseudo positive control in commercial IgM captureDengue ELISA and indirect IgM Dengue ELISA kits where the response iscompared to positive and negative controls provided in the kit.Biotinylation of mouse monoclonal anti dengue IgG (Clone 13C11) 2.56 mgof 13C11 Monoclonal antibody (IgG fraction) to dengue antigen (PanBioLtd, Windsor, Qld; product 13C7001) was equilibrated in iml 50 mM sodiumbicarbonate buffer, pH 8.0. To this was added 7511 freshly preparedEZ-Link™ Sulfo-NHS-LC-Biotin solution (1 mg/ml in water) (PierceChemical Company, Rockford, Ill.; product code 21335), and incubated atroom temperature for 1 hour. After the sample had undergone extensivedialysis against PBS (phosphate buffered saline, final sample volume 1.2ml), the protein concentration was estimated by absorbance at 280 nm tobe 1.6 mg/ml.

The success of the biotinylation reaction was confirmed by ELISAin whichdilutions of the biotinylated 13C11 antibody was added to wellscontaining immobilised-dengue antigen. Non biotinylated 13C11 was usedas a negative control. A streptavidin-horse radish peroxidase conjugatewas used to visualise the presence of the biotinylated 13C11 antibody.

Dengue IgM Indirect EHSA

The reagents used in this assay were as follows:

ELISA plate coated with dengue 2 antigen (PanBio Pty Ltd, Windsor Old)

-   -   IgNI Positive Control Serum, IgM Cult-off Calibrator Serum,        Negative Control Serum for IgM (PanBio Pty Ltd provided in the        kit)    -   Biotinylated mouse anti dengue IgG (clone 13C11),        concentration=1.6 mg/mil (biotinylation was performed as        described above).    -   Non biotinylated mouse anti dengue IgG (clone 13C11),        concentration=1.6 mg/ml    -   Control Linker Reagent: Strep-C_(H)3μ Preparation-A,        concentration 0.17 mg/ml.

Control linker reagent was mixed with biotinylated and non-biotinylated13C11 Nlab prior to the assay in the following proportions:

-   1. 13C11-Biotin ( 1/10,000)+Control Linker Prep A 1/10-   2. 13C11-Biotin ( 1/10,000)+Control Linker Prep A 1/100-   3. 13C11-Biotini ( 1/10,000)+Control Linker Prep A 1/1000-   4. 13C11 ( 1/10,000) [nonbiotinylated]+Control Linker Prep A 1/10-   5. 13C11 ( 1/10,000) [nonbiotinylated]+Control Linker Prep A 1/100-   6. 13C11 ( 1/10,000) [nonbiotinylated]+Control Linker Prep A 1/1000

Dilution was in serum diluent (Tris buffered saline with preservativesand additives) as supplied in the kit. Mixing took place at roomtemperature for 10 min with rotation.

Additions to ELISA plate were in the following order:

1. Samples as above, blank, positive, negative and cut-off serumcontrols (100 μl) incubated at 37° C. for 1 hour, followed by 6 washeswith diluted wash buffer.

2. HRP-labelled sheep anti Human IgM (Silenus/AvRAD; Code MAH) 100 μl of1/1000 dilution; incubated for 1 hour at 37° C. followed by 6 washeswith diluted wash buffer.

3. Reaction was developed with 100 μl TNB reagent 10 min at roomtemperature, followed by the addition of 100 μl 1M phosphoric acid.Samples were read at 450 nm.

The results obtained from this indirect assay are shown in Table 7.TABLE 7 Sample A450 Blank (Serum diluent only) 0.058 Negative serumControl 1/100 0.063 Positive Serum Control 1/100 1.419 Positive Cut-Off1/100 0.522 Positive Cut-Off 1/100 (duplicate) 0.580 Control Linker1/10 + 13C11-biotin 1.186 Control Linker 1/10 + 13C11-biotin (dup) 1.109Control Linker 1/100 + 13C11-biotin 1.070 Control Linker 1/100 +13C11-biotin (dup) 1.076 Control Linker 1/1000 + 13C11-biotin 0.212Control Linker 1/1000 + 13C11-biotin (dup) 0.183 Control Linker 1/10 +13C11 0.151 Control Linker 1/100 + 13C11 0.059 Control Linker 1/1000 +13C11 0.074

These results show tha the complex formed between the Strep-C_(H)3μlinker reagent and the biotinylated mouse anti dengue IgG (13C11) actedas a human positive control up to a dilution of at least a/100. Noreaction was observed if the mouse monoclonal IgG was not biotinylated,or if there was insufficient control linker reagent to capture thebiotinylated mouse Nlab.

Use of Control Reagent Streptavidin-C_(H)3μ in PanBio Dengue IgM CaptureELISA

The Dengue IgM capture ELISA test kit from PanBio Ltd (Windsor, Qld; CatNo DEMF-200) was used for this demonstration. The positive and cut-offcontrol sera provided contain human IgM antibodies to dengue. Thenegative control serum contains human IgM antibodies, but with nospecificity for dengue antigens.

Two test samples of the Streptavidin-Human C_(H)3μ domain linker reagentwere used:

-   -   a) Preparation A, 0.17 mg/ml    -   b) Preparation B, 0.10 mg/ml

These preparations were mixed with biotinylated and non-biotinylated13C11 Mab as described below.

The following samples were prepared for ELISA:

-   1. Blank—100 μl serum diluent only, provided in PanBio kit-   2. Negative Control Seruin-fromi PanBio kit; 100 μl, diluted 1/100    in serum diluent.-   3. IgNI Positive Cdontrol Seruin from PanBio kit; 100 μl, diluted    1/100 in serum diluent.-   4. Cut-off Calibrator from PanBio kit; 100 μl, diluted 1/100 in    serum diluent.-   5. Strep-C_(H)3μ Preparation A+13C11-Biotin: 10 μl of prepA diluted    to 1 ml with serum diluent to which 1 μl 13C11-Biotin ( 1/1000    dilution) was added (Final concentration of PrepA protein=1.7 μg/ml;    13C11-Biotin=1.6 μg/ml).-   6. StreP-C_(H)3μ Preparation B+13C11-Biotin: 10 μl of prepB diluted    to 1 ml with serum diluent to which 1 μl 13C11-Biotin ( 1/1000    dilution) was added (Final concentration of PrepB protein=1.0 μg/ml;    13C11-Biotin=1.6 μg/ml).-   7. Blank+13C11-Biotin (negative control) 1 μl 13C11-Biotin was added    to 1 ml serum diluent (Final concentration of 13C11 biotin=1.6-   8. Strep-C_(H)3μ Preparation A+13C11(non biotinylated) (negative    control): 10 μl of prepA diluted to 1 ml with serum diluent to which    1 μl 13C11 ( 1/1000 dilution) was added (Final concentration of    PrepA protein=1.7 μg/ml; 13C11=1.6 μg/ml).-   9. Strep-C_(H)3μ Preparation B+13C11 (non biotinylated) (negative    control): 10 μl of prepB diluted to 1 ml with serum diluent to which    1 μl 13C11 ( 1/1000 dilution) was added (Final concentration of    PrepB protein=1.0 μg/ml; 13C11=1.6 μg/ml).-   10. Blank+13C11 (negative control) 1 μl 13C11-Biotin was added to 1    ml serum diluent (Final concentration of 13C11 biotin=1.6 μg/ml).

Each sample was mixed on a rotating wheel for 10 min at roomtemperature, then 100 μl of each (some in duplicate) were added to ELISAstrips from the test kit which were pie-coated with polyclonal sheepanti human IgM. The strips were covered and incubated at 37° C. for 60min. then washed three times for 2 min with PBS containing 0.05% Tween20. At the same time as the above incubation, 2 ml conjugated monoclonalantibody tracer (PanBio: anti dengue-HRP) was added to one vial oflyophilised dengue anti-en (serogroups 1-4) and rocked gently at roomtemperature to aid in the dissolution of the dengue antigen. After theabove washes, 100 μl of the HRP conjugate was added to each well,incubated for 60 nun at 37° C. then washed three times for 2 min withPBS-0.05% Tween 20. 100 μl of TMIB reagent (3′,3′,5′,5′tetramethylbenzidine/hydrogen peroxide; supplied with the kit) was thenadded to each well and the strips incubated at room temperature for 10mins. The reaction was stopped by the addition of 100 μl 1M phosphoricacid and the colour intensity read at 450 nm. Results of this assay areshown in Table 8. TABLE 8 Sample A450 Blank (serum diluent only) 0.115Negative serum control 0.121 IgM positive serum control 2.678 PositiveCut-off Calibrator 1.198 Positive Cut-off Calibrator (duplicate) 1.235Preparation A + 13C11-biotin 1.914 Preparation A + 13C11-biotin(duplicate) 1.850 Preparation B + 13C11-biotin 1.200 Preparation B +13C11-biotin (duplicate) 1.344 Blank + 13C11-biotin 0.115 Blank +13C11-biotin (duplicate) 0.128 Preparation A + 13C11(non biotinylated)0.142 Preparation A + 13C11(non biotinylated) 0.126 Preparation B +13C11(non biotinylated) 0.113 Preparation B + 13C11(non biotinylated)0.124 Blank + 13C11(non biotinylated) 0.106

Both preparations of Strep-C_(H)3μ gave positive reactions in the ELISAat levels sufficient for the complex with mouse IgG to be used as areplacement for the positive control serum. The lower reading withpreparation B can be partly attributed to the lower concentration of theproduct. Results with the controls indicated that there were nosignificant background problems.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

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1-90. (canceled)
 91. A method for detecting presence of an antibody in abiological sample obtained from a test species, the method comprising:a) performing an antibody based diagnostic test on the biologicalsample; b) performing the antibody based diagnostic test on a positivecontrol, wherein the positive control shows a positive reaction; and c)comparing results of step a) with results of step b), whereby thepresence of an antibody in the biological sample is detected by apositive reaction in step a); wherein the positive control comprises acomplex formed between (i) an antibody or biologically active fragmentthereof from a first species, wherein the first species is differentfrom the test species and (ii) a bifunctional molecule, the bifunctionalmolecule comprising a binding region that binds to the antibody of thefirst species, and a constant region from an antibody of the testspecies, the constant region comprising at least one CH domain or anepitope thereof.
 92. The method according to claim 91, wherein thebifunctional molecule binds to the constant region of the antibody ofthe first species.
 93. The method according to claim 91, wherein thebinding region of the bifunctional molecule is of non-antibody origin.94. The method according to claim 93, wherein the binding region is aprotein selected from the group consisting of Streptococcal protein G,Staphylococcal aureus protein A, and Peptostreptococcus magnus proteinL, or a fragment thereof.
 95. The method according to claim 93, whereinthe binding region comprises fragment B of Staphylococcus aureus proteinA.
 96. The method according to claim 93, wherein the binding regioncomprises a mouse Fc y receptor or fragment thereof.
 97. The methodaccording to claim 93, wherein the binding region comprises histidinerich glycoprotein.
 98. The method according to claim 91, wherein thebinding region has a K_(D) for the antibody of the first species of lessthan 10⁻⁶ M.
 99. The method according to claim 91, wherein the bindingregion has a K_(D) for the antibody of the first species of less than10⁻⁸ M.
 100. The method according to claim 91, wherein the bindingregion binds to one or more non-naturally occurring group(s) provided onthe constant region of the antibody of the first species.
 101. Themethod according to claim 100, wherein the group(s) is a biotin moleculeand the binding region comprises streptavidin or a fragment thereof.102. The method according to claim 91, wherein the constant region fromthe antibody of the test species comprises one or more constant domainsfrom an IgM antibody.
 103. The method according to claim 102, whereinthe constant region from the antibody of the test species comprises oneor more C_(H)3μ domains.
 104. The method according to claim 91, whereinthe constant region from the antibody of the test species comprises oneor more constant domains from an IgG antibody.
 105. The method accordingto claim 104, wherein the constant region from the antibody of the testspecies comprises one or more C_(H)3γ domains.
 106. The method accordingto claim 91, wherein the constant region from the antibody of the testspecies comprises one or more constant domains from an IgA antibody.107. The method according to claim 91, wherein the constant region fromthe antibody of the test species comprises or consists of anon-naturally occurring combination of immunoglobulin C_(H) domains orepitopes thereof.
 108. The method according to claim 91, wherein theconstant region from an antibody of the test species consists of asingle C_(H) domain.
 109. The method according to claim 91, wherein thebinding region and the constant region from the antibody of the testspecies are linked directly or are separated by a linker molecule ofbetween 1 and 20 amino acids in length.
 110. The method according toclaim 91, wherein the first species is a rat or mouse.
 111. The methodaccording to claim 91, wherein the test species is a human.
 112. Themethod according to claim 91, wherein the antibodies to be detected areantibodies characteristic of a disease selected from the groupconsisting of dengue fever, Japanese encephalitis, rubella, spottedfever, herpes infection, parvovirus infection, melioidosis, typohid,leptospirosis, malaria, yellow fever, whooping cough, systemiccandidiasis/thrush, chicken pox, shingles, ADS, hepatitis, liver cancer,cervical cancer, infectious mononucleosis, nasopharyngeal carcinoma,Ross River fever, brucella, exanthum subitum (sixth disease/roseolainfantum), erythema infectiosum (fifth disease), Q fever, cold sores,genital herpes, spotted fever and scrub typhus.
 113. The methodaccording to claim 91, wherein the antibody based diagnostic test isselected from the group consisting of ELISA, immunochromatography,particle agglutination ELISA and biosensor assays.